Recombinant DNA technology refers generally to the technique of integrating genetic information from a donor source into vectors for subsequent processing. This typically entails introducing exogenous DNA into a host, whereby the transferred genetic information is copied and/or expressed in the new environment. Commonly, the genetic information exists in the form of complementary DNA (cDNA) derived from messenger RNA (mRNA) coding for a directed protein product. The vector is frequently a plasmid having the capacity to incorporate cDNA for subsequent replication in a host and, in some cases, actually to control expression of the cDNA and direct the host to make the encoded product.
This technology has progressed extremely rapidly in recent years, and a variety of exogenous proteins has been expressed in a variety of hosts. By way of example, some of the eukaryotic proteins to produced include: proinsulin (Naber, S. et al., Gene 21: 95-104 [1983]); interferons (Simon, L. et al., Proc. Nat. Acad. Sci. U.S.A., 80: 2059-2062 [1983] and Derynck, R. et al., Nucl. Acids Res. 1: 1819-1837 [1983]); and growth hormone (Goeddel, D., et al, Nature 281: 544-548 [1979]). (These publications and other herein referenced materials have been included to provide additional details on the background of the pertinent art and, in particular instances, the practice of invention, and are all incorporated herein by reference.)
It is now generally accepted that the mammalian immune response is mediated by a series of complex cellular interactions, coined the "immune network". The immune response had been viewed as comprising two different activities: a humoral and a cellular response. The humoral response is thought to consist primarily of the actions of soluble proteins, known as antibodies or immunoglobulins. The proteins have the capability of binding to, and assisting in the removal from body fluids of, matter perceived foreign (i.e., "non-self") through the recognition of antigenic sites on the foreign matter (known as an antigen). The cellular response is thought to consist of (as the name implies) cell mediated activities, for example macrophage phagocytosis and lymphocyte involvement in delayed-type hypersensitivity and allograft reactions. However, extensive research has proven that while humoral and cellular immunity individually may represent the final stages of an immune response, the entire response revolves around a series of very complex, network-like interactions of lymphocytes, macrophages and other cells acting in concert with each other and with immunoglobulins. Moreover, immunologists now hold the opinion that other soluble proteins (e.g., the so-called lymphokines produced by lymphocytes) play a critical role in controlling the sequence of events making up immunity.
Lymphokines apparently mediate cellular activities in a variety of ways. They have been shown to have the ability to support the proliferation and growth of various lymphocytes and, indeed, are thought to play a crucial role in the initial differention of pluripotential hematopoietic stem cells into the vast number of progenitors of the tremendously diverse immunologic cellular lineages. Cell lineages thought to be controlled in part lymphokines include two types of lymphocytes: B cells that can differentiate to produce and secrete the five major classes of immunoglobulins (.alpha., .delta., .gamma., .epsilon., and .mu.; known as the IgA, IgD, IgG, IgE, and IgM isotypes, respectively), and T cells of various subsets that through various means induce or suppress B cells and some of the other cells (including other T cells) making up the the immune network.
Another important cell lineage whose growth seems to be under partial control of lymphokines is the mast cell--a granule-containing connective tissue cell located proximate to capillaries throughout the body, with especially high concentrations in the lungs, skin, gastrointestinal and genitourinary tracts. Mast cells play a central role in allergy-related disorders, particularly anaphylaxis. Briefly stated, once certain antigens crosslink IgE class immunoglobulins bound to receptors on the mast cell surface, the mast cell degranulates and releases mediators (e.g., histamine, serotonin, heparin, kinins, etc.) which can cause anaphylactic and some other allergic reactions.
The inhibition of mast cell degranulation by blocking IgE binding has been reported (U.S. Pat. No. 4,161,522), but attempts to duplicate the experiments have been unsuccessful (Bennich, H. et al. Int. Archs. Allergy Appl. Immun. 53: 459-468 [1977]), although experimentation continues. Recently, however, clinical research to better understand (and thus potentially treat therapeutically) allergies, anaphylaxis, and other IgE related immune disorders has focused more on the mechanisms of IgE formation.
Ontogenetic studies of lymphocytes have demonstrated the presence of B cells in fetal liver and spleen. In rodents, B cells initially exhibit surface IgM, but shortly after birth a portion bear surface IgE. The differentiation of fetal B cells to IgE bearing cells appears to be independent of any antigen or T cells. However, the subsequent differentiation of IgE bearing cells to IgE secreting cells seems dependent on exposure to the antigen against which the B cell can make specific antibodies and to which a set of T cells can also bind (see generally, Ishizaka, K., Annals of Allergy 48: 320-324 [1982]). (This process may occur for other isotypes as well.)
Therefore, with no practical, readily apparent method known for halting the post-natal development of IgE bearing B cells, physicians have attempted to regulate the 0 response by antigen-specific inactivation of IgE bearing B cells or the associated T cells. This has entailed injecting modified antigens into allergic patients, a procedure which in some cases risks anaphylactic shock or reduction of other needed immune response capability. Moreover, many people develop allergy to a variety of different antigens, which would require a multitude of modified antigen preparations and injections. For these and other reasons, antigen specific inactivation has been at best moderately successful, but research work is continuing.
Within the last few years, a few research groups have reported the existence of soluble, lymphocyte-secreted, regulatory factors that appear to act selectively on the IgE response, but which are not antigen-specific. These factors have been only partially characterized, with three research groups reporting molecular weights ranging from about 15,000 to 60,000 to 200,000 daltons. (See, Katz, D. Immunology 41: 1-24 [1980]). At present, the relationship (or lack thereof) among the factors is not known with certainty, although they appear to be antigen non-specific and either suppressive or enhancing with respect to the IgE response. Unfortunately, research to partly characterize these factors has been hampered by a lack of sufficient quantities of factor to conduct extnesive protein analysis, as well as by assay procedures that are difficult, thime consuming and, in some cases, potentially subjective.
In spite of these drawbacks, research has progressed, albeit more slowly than if the materials were available in bulk. The best characterized factors are the so-called IgE binding factors (IgE-BF), which--as their name implies--have affinity for IgE. They are postulated to function by first binding to IgE-bearing B cells via surface IgE, and then through a complex, poorly understood series of events, to affect the differentiation of the B cells to IgE secreting plasma cells. Apparently, as IgE-BF's do not bind to other immunoglobulin bearing B cells, the factors are isotype specific.
At least two measurably active types of IgE-BF have been defined. These two factors have very similar molecular weights (about 15,000 daltons) and similar affinity for IgE. They seem to differ primarily in their carbohydrate moieties, with one, IgE-potentiating factor, probably containing N-linked mannose-rich oligosaccharide and having terminal sialic acid; whereas the second, IgE-suppressor factor, is probably O-glycosydically linked and has a terminal galactose sugar. Interestingly, it seems that one T cell set has the capacity to form either factor, the particular factor produced (or perhaps another IgE-BF with no detectable IgE suppressor or potentiating activity) being dependent on the cell's environment. (See generally, Ishizaka, K., Lymphokines 8: 41-80 [1983].)
The research described immediately above has opened new and exciting vistas in the potential control of IgE levels. However, a full investigation of the proposed relationship between the two binding factors and their activities will probably necessitate ascertaining additional structural data, e.g., substantially complete sequence analysis of the molecules in question. Protein sequencing offers, of course, a possible means to solve the problem, but it is very difficult work experimentally, especially on small quantities of material, and to date has not proved useful. Having the capability of making bulk quantities of polypeptides exhibiting mammalian IgE-BF activity is probably essential to understand the factors' modes of action and may further serve to facilitate greatly the study of the biology of isotype regulation in general. Accurate and complete sequence data on a rodent IgE-BF will also aid in the search for human IgE-BF proteins, enhancing the likelihood of treatment for IgE mediated diseases. Finally, additional information on any lymphokine will assist in evaluating the roles of the various factors and cells of the immune network, providing insight into the entire immune system - with the concomitant therapeutic benefits.
Thus, there exists a significant need for extensive nucleotide sequence data on the DNAs coding for, and amino acid sequences of, proteins exhibiting IgE-BF activity, as well as a simple and economic method of making substantial quantities of such material. The present invention fulfills these needs.